Isolation and genotypic characterization of extended-spectrum beta-lactamase-producing Escherichia coli O157:H7 and Aeromonas hydrophila from selected freshwater sources in Southwest Nigeria

The proliferation of antibiotic-resistant bacteria and antimicrobial resistance is a pressing public health challenge because of their possible transfer to humans via contact with polluted water sources. In this study, three freshwater resources were assessed for important physicochemical characteristics as well as heterotrophic and coliform bacteria and as potential reservoirs for extended-spectrum beta-lactamase (ESBL) strains. The physicochemical characteristics ranged from 7.0 to 8.3; 25 to 30 °C, 0.4 to 93 mg/L, 0.53 to 8.80 mg/L and 53 to 240 mg/L for pH, temperature, dissolved oxygen (DO), biological oxygen demand (BOD5) and total dissolved solids, respectively. The physicochemical characteristics mostly align with guidelines except for the DO and BOD5 in some instances. Seventy-six (76) Aeromonas hydrophila and 65 Escherichia coli O157: H7 isolates were identified by preliminary biochemical analysis and PCR from the three sites. Among these, A. hydrophila displayed higher frequencies of antimicrobial resistance, with all 76 (100%) isolates completely resistant to cefuroxime and cefotaxime and with MARI ≥ 0.61. The test isolates showed more than 80% resistance against five of the ten test antimicrobials, with resistance against cefixime, a cephalosporin antibiotic being the highest at 95% (134/141). The frequency of the detection of the resistance genes in the A. hydrophila isolates generally ranged between 0% (blaSHV) and 26.3% (blaCTX-M), while the frequency of detection among the E. coli O157:H7 isolates ranged between 4.6% (blaCTX-M) and 58.4% (blaTEM). Our findings indicate that the distribution of antibiotic-resistant bacteria with diverse ESBL-producing capabilities and virulence genes in freshwater sources potentially threatens public health and the environment.


Materials and methods
Study area description. Initial reconnaissance survey and mapping of the study area were done after the State Ministries of Environment, Water Resources, Lands, and Housing had been duly consulted. The study sites included three rivers (Erinle River, Asejire River and Esimirin Rivers) which fall within three local governments areas of Osun State in the South-western geopolitical zone of Nigeria with GPS coordinates 7°25′44″ N, 4°13′14″ E; 7°21′40″ N, 4°11′00″ E and 7°44′44″ N; 4°29′22″ E respectively (Fig. 1). Factors considered in the selection of the study sites include anthropogenic activities, domestic, agricultural, and industrial activities along the watercourse, sanitary states and the economic importance of the rivers. For ease of reference, the study sites are coded as River-A: Erinle River, River-B: Asejire River and River-C: Esimirin River, while the descriptions of the study sites are depicted in Table 1.
Water sampling for physicochemical parameters. Water samples for the physicochemical characteristics of the rivers were collected fortnightly from each of the study sites over a six-month sampling period. Some critical physicochemical characteristics were determined in situ. These include the use of Crison Multimeter MM40 (Crison Instrument S.A., Barcelona, Spain) for water pH, temperature (temp °C), total dissolved solids (TDS mg/L), and turbidity by microprocessor turbidity meter (HACH Company, model 2100P). Dissolved oxygen (DO) was determined by HQ40d BOD meter (HACH Company, Loveland, C, USA), while the biological oxygen demand (BOD 5 ) was determined in the laboratory after five days of incubation in a dark cupboard 24 . All analyses were done in triplicates, and values were reported as means ± standard error of means.
Sample collection and microbiological analysis. One hundred and eight (108) river water samples were aseptically collected in sterile 1 L glass bottles and transported in cooler boxes (4 °C), and processed upon arrival at the Microbiology laboratory (Department of Microbiology, Obafemi Awolowo University, Nigeria). Samples were collected fortnightly over six months, divided into the raining (July to September) and the dry (October to December) seasons. The collected samples were subjected to bacteriological analysis to determine heterotrophic plate count (HPC), total coliforms (TC), faecal coliforms (FC), A. hydrophila, and E. coli O157:H7 counts. Enumeration of the presumptive bacteria was done by making appropriate serial dilution of the samples and spread plating 100 µL aliquots of the diluted samples on appropriate media in triplicate. All media used for Antibiotic susceptibility profiling of isolates. Antibiotic susceptibility patterns of A. hydrophila and E. coli O157:H7 were assayed using the disc diffusion method on freshly prepared Muller Hinton Agar plates, and the results were interpreted according to the Clinical and Laboratory Standard Institute 25 . The susceptibility profiles of the isolates were established against a panel of nine commercially available antibiotics. The antibiotics used in the study include amoxicillin (25 µg); augmentin (10 µg); cefixime (5 µg); cefotaxime (30 µg); ceftazidime (30 µg); cefuroxime (30 µg); ciprofloxacin (5 µg); gentamicin (10 µg); nitrofurantoin (30 µg) and ofloxacin (5 µg).

Determination of isolates multiple antibiotic resistance indices (MARI). The MARIs of isolates
were estimated based on their phenotypic antibiotic resistance patterns 26 . Multiple antibiotic resistance (MAR) was defined in the study as resistance to three or more test antibiotics. The formula used for the MARI estimation is as follows: Phenotypic detection of ESBL production in A. hydrophila and E. coli O157:H7 isolates. The double-disc synergy test was employed to characterise the phenotypic production of ESBL, according to CLSI (2018). Briefly, freshly grown cultures were standardized (0.5 McFarland) and spread evenly to form a lawn culture on Muller Hinton agar plates. An antibiotic disc containing amoxicillin/clavulanic (20/10 µg) acid was carefully placed at the centre of the plates while two discs of third-generation cephalosporin (e.g., ceftriaxone, cefotaxime or ceftazidime) antibiotics were carefully placed at either 15 mm or 20 mm apart respectively, centreto-centre to that of the amoxicillin/clavulanic acid disc. The plates were incubated inverted at 37 °C for 24 h. The positive result for ESBL production was taken as any distortion or increase in the zone toward the amoxicillin/ clavulanic disc.

Molecular characterisation of ESBL producing genes of isolates.
Total genomic DNA was extracted from freshly grown cultures by boiling method 27 . The DNA extracts were stored on ice and used for PCR immediately to detect ESBL-producing genes, including bla CTX-M , bla SHV, and bla TEM . Amplifications were carried out in a final reaction volume of 25 µL consisting of 12.5 µL 2X PCR Master Mix Thermo Scientific Inc., Waltham, MA, USA), 1.0 µL each of forward and reverse primers (10 pmol), 5.5 µL of nuclease-free water and 5.0 µL of DNA template. The specific primers and the annealing temperatures are shown in Table 2. The amplicons were resolved on 1.5% agarose gel electrophoresis stained with 0.5 mg/L ethidium bromide with 0.5 × TBE buffer and visualized under the wave ultraviolet transilluminator and photographed using gene gel bioimaging system. Statistical analysis. The student t-test was used to compare the means of physicochemical and microbiological parameters for the two seasons. One-way analysis of variance and Pearson correlation test was used to compare the relationship between physicochemical and microbiological parameters for the three sites. The confidence limit was set at p ≤ 0.05 for all analyses.

Results
Physicochemical parameters. The physicochemical characteristics of the three rivers assessed in this study are presented in Table 3, and the seasonal comparison of the characteristic is in Table 4. The data shows the mean values of triplicate determinations ± the standard error. The parameters evaluated were generally compared to the stipulated standards for drinking water by the Standard Organisation of Nigeria 34 and the World Health Organisation 35 international standards for drinking water ( Table 3).
The pH values determined at River-A varied between 7.0 and 7.3 during the raining season (July to September), while they varied between 7.2 and 7.8 during the dry season (October to December). Similarly, the pH at River-B varied between 7.0 and 7.5 during the raining season and 7.1 and 8.3 for the dry season, respectively. At River-C, the values obtained for pH ranged between 7.1-74 and 7.5-7.9 for raining and dry seasons. The evaluated pH values were statistically significant during the two seasons (Table 4) at p < 0.05, and the values generally fell within the stipulated limits for drinking water according to the SON and the WHO standards ( Table 3).
The temperature regimen obtained at all sampling points and across both seasons ranged between 25 and 32 °C. Temperature for most of the sampling period was within the WHO recommended limits of 25-30 °C except at River-A and River-C, where temperatures were slightly above the limits during the dry months of November and December (Table 3). However, the temperature values obtained were, statistically significant at p < 0.05 or p < 0.01. Likewise, total dissolved solids concentrations at all sampling points complied with the recommended limits of both the SON and WHO ( Table 3). The TDS values were statistically insignificant during the two seasons ( Table 4).
The determination of the dissolved oxygen in the river water samples showed ranges of 0.4-6.5 mg/L, 1.3-9.3 mg/L, and 0.9-6.2 mg/L at River-A, River-B, and River-C, respectively. However, over the sampling period, only 22.2% (24/108) of the water samples had DO concentrations higher or equal to 5 mg/L for drinking water, as recommended by the WHO ( Table 3). The statistical significance test for the DO showed that the values at the three sample sites do not vary significantly at p < 0.05. The BOD 5 values at the three sampling sites ranged between 0.53 and 8.80 mg/L throughout the sampling period. Similar to the DO values, the BOD 5 values do not vary significantly at p < 0.05.
The seasonal statistical analysis of the pH values showed no significant variations across the three study sites. Equally, other parameters, including temperature, DO, and BOD 5 , do not vary significantly (p < 0.05) seasonally at the three study sites except TDS, whose values varied significantly during the two seasons.
Bacteriological characteristics and abundance of the target microbial groups. As presented in Table 5, the prevalence and distribution of the target bacterial groups varied widely in the samples analysed. The one hundred and eight (108) river samples analysed had at least one of the target bacteria. Table 5 shows the presumptive counts of the target bacteria generally ranging from 7.1 × 10 2 to 1.7 × 10 7 cfu/100 mL for heterotrophic plate counts; 1.4 × 10 2 to 8.3 × 10 4 cfu/100 mL for total coliforms; 1.0 × 10 2 to 3.1 × 10 4 cfu/100 mL for faecal coliforms; 1.1 × 10 2 to 1.4 × 10 4 cfu/100 mL for A. hydrophila and 1.0 × 10 2 to 4.2 × 10 5 cfu/100 mL for E. coli O157:H7.
The lowest HPC count was observed during the raining season in July 2018 at River-A, while HPC density was too numerous to count (TNTC) during the dry season at the same sites. For the coliforms, the lowest count of TC (1.4 × 10 2 cfu/100 mL) was observed during the raining months of July at River-B, while the lowest count of FC was observed at River-C during the raining season. The highest concentrations of TC (8.3 × 10 4 cfu/100 mL) and FC (3.1 × 10 4 cfu/100 mL) occurred during the dry season at River-C and River-B, respectively (Table 5). A. hydrophila had the least count of 1.1 × 10 2 cfu/100 mL at River-C in August 2018 while the highest density of A. hydrophila (1.4 × 10 4 cfu/100 mL) was recorded at River-B in November 2018. Presumptive E. coli O157:H7 was detected in all the samples analysed, with the lowest count (1.0 × 10 2 cfu/100 mL) occurring at River-B in July 2018 and the highest count in November, also at the same river.
Statistical comparison of the mean values from the three rivers across both seasons showed that the counts exceeded the standard limit of 0 cfu/100 5 mL of coliforms for portable and water as recommended by the WHO, the SON, and the FMEnv [34][35][36] . Also, the mean values of the microbial populations for the samples collected at all three sites during the dry months generally exceeded those collected during the raining months, with highly significant differences for microbiological parameters such as HPC (p = 0.0013), TC (p = 0.0019), FC (p = 0.0008) and A. hydrophila (p = 0.0012). Similarly, there is a slightly positive correlation between the physicochemical characteristics of the rivers and the microbiological qualities. For instance, increased temperatures, mostly observed during the dry months, appear to favour the microbial population densities of HPC, TC, FC, and A. hydrophila. Also, pH exhibited a strong positive correlation (r = 0.9219) with FC densities. However, an inverse correlation generally existed between the microbial density and dissolved oxygen in the samples.

Molecular characterization of antibiotic resistance and virulence genes in isolates.
The genotypic characterization of the isolates by PCR assays revealed the presence of the three ESBL-producing genes selected for this study, as shown in Table 7. The frequency of the detection of the resistance genes in the A. hydrophila isolates generally ranged between 0% (bla SHV ) and 26.3% (bla CTX-M ), while the frequency of detection among the E. coli O157:H7 isolates ranged between 4.6% (bla CTX-M ) and 58.4% (bla TEM ). Some of the representative gel electrophoretic images of the detected genes are shown in Supplemental Fig. 1a-

Discussion
Rivers are waterways of strategic importance for the sustenance of life and economic growth across the globe. Indiscriminate pollution and declining freshwater quality threaten human health and economic development and is a cause for concern. Proper maintenance of a healthy aquatic ecosystem depends on the physicochemical characteristics and biological diversity, which have interdependent effects on each other 37 . Regular and proper monitoring of waterbodies will not only assist in preventing disease outbreaks but also forestall further water quality deterioration towards human health and environmental protection.
Even though the water samples analysed in this study complied with the recommended standards in terms of the physicochemical characteristics and for most of the sampling period, there were certain instances where the water quality did not comply with the set limits. For instance, as depicted in Table 4, only approximately 22.2% of the water samples analysed had dissolved oxygen within the recommended limit set by the WHO (Table 3). Dissolved oxygen is a vital component that determines the quality of a waterbody and the trophodynamics of an  www.nature.com/scientificreports/ aquatic ecosystem. The concentration of dissolved oxygen close to saturation implies a relatively healthy water system, while the United States Environmental Protection Agency (U.S. EPA) recommends an oxygen concentration exceeding the chronic criterion for growth (4.8 mg/L) for a healthy water ecosystem 38 . Low dissolved oxygen levels below the criterion may lead to a deleterious effect on the health of the aquatic ecosystem, such as anoxia or hypoxia conditions, which in turn may result in the alteration of the ecosystem balance. Apart from anthropogenic influences, other factors, such as atmosphere-water surface exchange, nitrification, sediment-water exchange, mineralisation, respiration, and photosynthesis, affect the dissolved oxygen dynamics of an aquatic system 39 . The relatively low dissolved oxygen observed in some samples may be due to the oxygen-demanding substances. The dissolved oxygen regime observed in the study is similar to those reported elsewhere 40,41 . Biological oxygen demand assessment is another useful characteristic in measuring the compliance of water systems with set limits and the estimation of dissolved or biodegradable organics that may deplete oxygen 42 . The BOD 5 regime of the samples was mostly in line with the recommended limits or ≤ 6 mg/L except on a few occasions ( Table 4). The non-compliance of the river water to the limits in some instances may be due to the release of some oxygen-demanding substances into the river, which may be due to numerous small-scale industrial activities around the river courses.
Enteric bacteria are some of the mostly encountered microorganisms in aquatic systems. This study investigated the abundance of enteric bacteria and some important gastroenteritis pathogens in the water samples from three rivers. More than 97% of the water samples analysed in this study had HPC counts higher than the WHO standard of 100 cfu/ml (Table 5), while significantly higher bacterial counts were generally observed during the drier months than the raining months. HPC or standard plate count includes all bacteria, mould, and yeast that require organic carbon source and certain inorganic salts for their growth. Although some researchers have divided opinions on the importance of HPC measurement in water systems, it is generally believed that increases in HPC bacteria may be an early indicator of pollution. The progressive rise of HPC may have implications for public health, particularly among immunocompromised individuals and may signal the likely presence of opportunistic pathogens, which may cause gastroenteritis, diarrhoea and other related intestinal infections 43 . Our results are comparable to those reported elsewhere 44 .
Total coliforms (TC) are a broad range of collectively harmless bacteria found mainly in the gastrointestinal tract of humans, other warm-blooded animals, and the environment. A particular subset of the group is the faecal coliforms (FC), with the most frequent member being Escherichia coli. The FCs are mainly separated from other coliforms by their ability to grow at elevated temperatures (44.5 °C) with or without oxygen. Similar to the trend observed for HPC, coliform bacteria counts were generally higher during the dry months of October to December, as seen in Table 5. The highest count of TC (8.3 × 10 4 cfu/100 mL) was observed at River-C in December, while the lowest count occurred at River-B in July. The highest density of FC (3.1 × 10 4 cfu/100 mL) was recorded in November at River-B, while the least count was in July '18 at River-C (Table 5). Although coliforms themselves are generally not pathogenic, their presence indicates faecal contamination of water by human or other warm-blooded animals' faecal matter. Their presence also indicates the possible presence of pathogens and other parasites, which may be associated with diseases such as typhoid fever, viral and bacterial gastroenteritis, and hepatitis A, among others 45 . Faecal contamination of water presents a potential risk for human exposure. As observed in our results, coliform bacteria might have resulted from the overflow of domestic sewage and other nonpoint sources of anthropogenic and agricultural waste materials. Counts observed in this study mostly complied with the WHO limit of 10 3 cfu/100 mL during the raining months, which may be a result of the dilution effect of rain around this time of the year, while counts were generally higher than the limits during the drier months. Our findings are similar to those reported elsewhere 44,46 .
Motile Aeromonas species are opportunistic pathogens isolated frequently from polluted water sources with different degrees of contamination. However, various reports have identified Aeromonas as an aetiological agent of human enteric infections [47][48][49] . Typical faecal indicator bacteria monitoring may not adequately assess the public health risk associated with opportunistic pathogens such as Aeromonas. In the current study, Aeromonas spp. populations at the 3 study sites ranged between 1.2 × 10 2 -9.8 × 10 3 cfu/100 mL at River-A, 3.3 × 10 2 -1.4 × 10 4 cfu/100 mL at River-B and 1.1 × 10 2 -7.4 × 10 3 cfu/100 mL at River-C. Wide variations were generally observed in the distribution of Aeromonas spp. in the water samples, with densities mostly increasing in the drier months as compared to the raining months (Table 5). Reduced rainfalls and more stable temperatures may contribute factors to the general increase in bacterial densities during the dry months 50,51 . Seventy-six (18.1%) of the 420 randomly picked isolates were identified as Aeromonas spp. by physiological and conventional biochemical analyses. According to the test, the 76 isolates were Gram-negative, motile, rod-shaped, glucosefermenting, and facultative anaerobes. The isolates were also oxidase, catalase, and indole positive while negative for the urease test (Table 6). Aeromonas spp. have become important emerging human pathogens causing wound infection, septicaemia and diarrhoeal illnesses 52,53 . The prevalence and distribution of Aeromonas species in aquatic systems, their roles as microbial pollutants for surface and drinking water supplies, and their ability to induce diseases are of great concern to public health.
Cattle are the main reservoirs of E. coli O157:H7, which they typically excrete at 10 2 to 10 5 colony-forming cfu per gram of faeces 54,55 . E. coli O157:H7 bacteria commonly exist in the intestinal tracts of cattle and may find their way into waterways through agricultural organic waste runoffs or direct deposition of faecal matter into surface waters. Surface water close to farms may thus represent a potential reservoir for enteric pathogens, including E. coli O157:H7, which may allow re-infection cycles and increasing the potential for the pathogens to spread 56 . E. coli O157:H7 was detected in all the samples collected from the three study sites selected for this study, with presumptive counts ranging from 2.7 × 10 3 to 1.5 × 10 4 cfu/100 mL, 1.0 × 10 2 to 1.4 × 10 4 cfu/100 mL and 2.3 × 10 3 to 4.2 × 10 5 cfu/100 mL at River-A, River-B, and River-C respectively. The detection of E. coli O157:H7 bacteria from all the study sites suggests that the rivers may serve as reservoirs for this pathogen and may be a significant source of dissemination of E. coli O147: H7 in the environment and subsequently to humans via direct contact  60 . The E. coli O157:H7 counts obtained at the various sites in this study did not correlate with coliform counts and generally fell short of the coliforms recommended limits, which suggests the unsuitability of the water sources for direct domestic use or other contact activities. As recorded in this study, high levels of microbiological pollution indicators were generally observed in samples from the three study sites, mostly during the dry seasons might have resulted from the intense anthropogenic activities around the study sites, as described in Table 1.
In the current study, the identified Aeromonas spp. and E. coli O157:H7 isolates subjected to antibiotic susceptibility testing (AST) showed increasing resistance towards 3 antibiotics (augmentin, 97.1%, cefotaxime, 100%, and cefuroxime, 100%) out of a panel of 10 conventional antibiotics used for the test (Table 6). Overall, the highest incidences of antibiotic resistance were observed against cefixime, with a frequency of 95% (134/141), while the least resistance was against ciprofloxacin, at a frequency of 19.9% (28/141). Antimicrobial resistance poses a serious global public health threat because of the continuous emergence and spread of multidrug-resistant "superbugs". The annual estimate of illnesses associated with antimicrobial-resistant pathogens is approximately 700,000 globally, while deaths associated with antimicrobial resistance have been projected to reach 10 million, with a global loss of about US$100 trillion by the year 2050 61,62 . Indiscriminate use of antimicrobials remains a significant factor contributing to the proliferation of antimicrobial resistance. Our finding contradicts the report of 63 , who reported 100% resistance against ciprofloxacin while researching the resistance profiles of Aeromonas isolates from water-related wound infections. Contrarily, our observation is corroborated by the report of Koksal et al. 64 , who reported only 1% resistance to ciprofloxacin for Aeromonas strains isolated from drinking water samples in Istanbul, Turkey.
The multiple antibiotic index (MARI) threshold of 0.2 is used to determine the possible exposure of isolates to antibiotic resistance selective pressures such as antimicrobial and heavy metals in their environment 26 . The observed MARIs in this study generally show Aeromonas spp. having MARI values (Table 6) above the threshold at all the sampling points, while E. coli O157 isolates were mostly below the threshold. This might have resulted from the occurrence of numerous Aeromonas isolates with high multidrug resistance phenotypes, as observed in the AST analysis in this study.
Extended-spectrum beta-lactamases (ESBLs) are important enzymes produced by bacteria which hydrolyze and inactivate beta-lactam antibiotics. Since their initial discovery in the 1980s, they have increased in prevalence among bacterial pathogens. The production of ESBL is a significant health concern because they reduce or destroy the potency of common drugs, including penicillins, carbapenems and cephalosporins, thus making infections more difficult to treat with increased complications 65 . In this study, a high prevalence (89.5%; 68/78) of phenotypic ESBL production was observed among the Aeromonas isolates from the water samples, and E. coli O157:H7 has a prevalence of 58.5% (38/65). ESBL-producing bacteria have increasingly been reported in clinical and environmental isolates in recent years, with three major classes of chromosomally-mediated β-lactamase-Amber class B, C and D being recognized 66,67 . The actual prevalence of ESBL-producing bacteria is believed to be underestimated because of the various challenges associated with their identification in clinical laboratories 68 . The prevalence of ESBLs in Africa has mostly been reported from human and animal samples (between 11-72%), while current reports from environmental samples (7-79%) are mostly from wastewater 69 . This current study reports ESBL prevalence in Aeromonas species and E. coli O157:H isolates recovered from freshwater sources which may present a health risk to the communities and persons in direct contact with water from the sources. Our observation is significant because of the scarcity of documented information on the prevalence of ESBL-producing Aeromonas species and E. coli O157:H7 in rivers in the southwest region of Nigeria. Our observation is similar to the study of Banu et al. 70 , who reported a prevalence of 98% (94/96) for ESBL-producing E. coli from river water samples in Ghana.
The molecular characterisation of the ESBL genotypes showed that most of the ESBL-producing Aeromonas spp. harboured the bla CTX-M gene, which was detected in 26.3% (20/76) isolates analysed by PCR, while the bla TEM gene was not detected in any of the Aeromonas isolates and 2.6% (2/76) harbouring the bla SHV gene (Table 7). In contrast, most of the ESBL-producing E. coli O157:H7 isolates harboured the bla TEM gene 58.5% (38/65), while the bla SHV gene was detected in 18.5% (12/65) of the isolates and bla CTX-M detected in 4.6% (3/65) isolates. The majority of molecular studies worldwide have reported the bla TEM gene as the most commonly encountered ESBL genotype [71][72][73] . This coincides with our observation among the E. coli O157:H7 analysed in this study; however, the bla TEM gene was not detected among the Aeromonas isolates from this study.
The bla TEM genes are among the most clinically relevant beta-lactamases due to their ability to produce enzymes with broad-spectrum activities that hydrolyse the cyclic amide bond of the beta-lactam rings, including those of penicillins and cephalosporins. The genes were initially thought to be chromosomal; however, they have been identified on plasmids, and their mobility has been associated with transposon and integrons 74 . Similarly, the bla SHV genes have become clinically important because of their ability to hydrolyse a broad spectrum of Scientific Reports | (2023) 13:10746 | https://doi.org/10.1038/s41598-023-38014-y www.nature.com/scientificreports/ antibiotics by causing changes to the configurations of the active sites of drugs, including monobactam and carbapenems. The group currently encompasses a large number of allelic variants which can be exchange between pathogens via horizontal gene transfer 75 . Mobile genetic elements (including plasmids and integrons) and epidemic strains of Enterobacteriaceae have been mostly associated with the widespread and success of the bla CTX-M genes. The mobile genetic elements are been identified to provide the conditions for the persistence of this gene among bacteria through the co-location of multiple resistance determinants and the co-selection by several antimicrobials 76 . The PCR amplification of virulence genes in the isolates revealed the presence of hylA, one of the genes involved in haemolysin production in 9 of the E. coli O157:H7 isolates. Enterohaemorrhagic E. coli (EHEC) is known to harbour other putative virulence genes, such as stx1 and stx2. Two genes (rfbE O157 and fliC H7 ) were also detected in 21 and 14 E. coli O157:H7 isolates. The gene rfbE O157 encodes the E. coli O157:H7 somatic antigen O157, while the fliC H7 encodes the structural flagella antigen designated H7. Both of these genes provide molecular identification of the O157:H7 E. coli serotype and can be used to differentiate the highly virulent serotype from E. coli strains 33 .
In conclusion, direct or indirect contact with polluted water sources can lead to an outbreak of infectious diseases contributing to the increased burden of enteric and extra-intestinal diseases. Attempts at providing safe, clean, and adequate water supplies will not safeguard human health and promote socioeconomic development and human dignity. Although the findings of this study showed that some physicochemical characteristics of the sampled rivers were within the acceptable limits, even so, those characteristics that do not comply with the set limits cannot be ignored. In the same vein, the bacteriological quality of the samples suggests potential human health and environmental risk. The detection of potentially pathogenic and multidrug-resistant Aeromonas and E. coli strains and high levels of indicator bacteria reflects the possible threat associated with direct contact or ingestion of water from these freshwater sources without adequate treatment. One major significant implication regarding the detection of the virulence and antimicrobial resistance genes in isolates from these freshwater sources is their possible persistence and spread to other potential pathogens not targeted in this study. Therefore, this necessitates effective intervention for managing and improving these important freshwater resources in the interest of public health and environmental protection.